See also Probiotics
Introduction:
The intestinal microbiota is confined most to the colon where some 1.5 kg of microbes or 1014 microorganisms reside. There are more genes in the human microbial than in the human genome. In the gut associated lymphoid tissue (GALT) enterocytes or intestinal epithelial cells represent the first barrier against invading organisms by secreting either mucin or defensins or sensing pathogens via TLRs. DCs also act as APCs. In aged GALT, a marked impairment of the immune response has been reported. Microbial components account for the production of short chain fatty acids (SCFA) such as butyrate, acetate and propionate. SCFA have antiinflammatory and anti-neoplasmic activities which exert a protective function. (Magrone, Immunity & Ageing 2013, 10: 31).
Bacteria living in the gut are being studies not only because they play a role in gastroinestinal disorders like inflammatory bowel disease, Crohn’s disease and colorectal cancer, but also because they can influence diverse and distal organs such as the brain. The gut-brain-axis begins with the enteric nervous system (ENS), a network of neurons that runs through the gastrointestinal tract. From there, the gut communicates with the brain via the vagus nerve, which connects the ENS to the central nervous system.
Probiotics: The core microbioa consists of bacteria beneficial for its host. When individual microbiota members are obtained in pure cultures and provided back to its host to improve its performance, such cultures are called probiotics. By definition, probiotics are live microorganisms that, when adminstiered in adequate amounts, confer a health benefit on the host. Their benefits are expressed at multiple levels. Probiotics may degrade feed ocmponents difficult for their host to digest into easily metabolized orgaic acids. Release of organic acids also decreases pH, which can suppress the epxression of virulence factors of pathogens such as Salmonella enterica. Probiotics may affect the rest of gut microbiota by production of antimicrobial substances. Probiotics can also modulate the immune resposne of their host or positively affect gut physiology associated with more efficient nutrient resorption. (Rychlik, “Ecological adaptations of gut microbiota members and their consequences for use as a new generation of probiotics” International J. Molecular Sciences, 2021).
Gut: is your gastrointestinal (GI) or digestive system, a long tube from your mouth to your anus responsible for digesting food, absorbing nutrients, and eliminating waste. It includes the mouth, esophagus, stomach, small and large intestines, and is supported by organs like the liver, pancreas, and gallbladder. A healthy gut also contains trillions of microbes, collectively called the gut microbiome, which are crucial for digestion, nutrient synthesis, and immune function.
Mucosal Epithelium: The architecture of the mucosal epithelium contains several barriers that attempt to prevent or impede infection by pathogenic bacteria. Mechanisms of protection are employed by all of these barriers in order to maintain the integrity of the epithelial cell monolayer and limit inflammation-associated damage. The luminal side of the intestinal epithelium is covered with a thick layer of mucus primarily composed of mucins, the main secretory product of goblet cells. Mucins are high molecular weight glycoproteins that aggregate to form a “gel-like” barrier to defend against endogenous or exogenous luminal insults. To date, at least 17 highly conserved mucins have been identified, each with varying specificities for different epithelial tissues. The mucosal layer consists of an inner layer of mucus that is firmly adherent to the intestinal epithelial cells (mainly comprised of cell surface mucins) and a looser outer layer of mucus (mainly comprised of secreted mucins). For quite some time, the mucus layer of epithelial surfaces was thought to solely serve the purpose of providing a physical barrier, preventing access of pathogenic bacteria or resident microbiota to the epithelial cells. However, it has been increasingly realized that the mucins in the outer sublayer of the mucosal barrier also provide an energy source for both resident microbiota and pathogenic microorganisms capable of adhering to the mucus layer. This layer provides both commensal and pathogenic microorganisms with a niche in which to grow and colonize the intestine. The inner layer of the mucosal surface
is considered “sterile,” largely due to the presence of antimicrobial peptides secreted by Paneth cells. The mammalian intestinal microflora contains ~10 to the14 resident bacteria, comprising ~1,000 species, and they reside in the outer sublayer of the mucosal barrier on the luminal side of the intestinal epithelium. Paneth cells are specialized epithelial cells located at the base mof crypts of Lieberkuhn that generate and secrete antimicrobial peptides of ~20–40 amino acids in length. There are four families of antimicrobial peptides: defensins, cathelicidins, histatins, and lactoferrin. Defensins are positively charged and directly interact with the negatively charged membrane of pathogenic microorganisms resulting in membrane destabilization and pore formation. Cathelicidins are also positively charged, and they function in binding and neutralizing lipopolysaccharides (LPS), ultimately resulting in pore formation. Unlike defensins and cathelicidins, histatins do not interact with the membranes of pathogenic bacteria. Instead,
histatins are ingested by the bacteria, inhibit mitochondrial respiration, and kill the microorganism by generating reactive oxygen species. The lamina propria is the connective tissue underlying the epithelial cell monolayer. It contains multiple immune effector cells including B cells, T-cells, dendritic cells, natural killer (NK) cells, macrophages, eosinophils, and mast cells. If enteric pathogens are capable of surmounting the barriers described above and penetrate the intestinal epithelium, a coordinated immune response utilizing these immune effector cells is activated. The most abundant B cell found in the lamina propria is the IgA-secreting B cell. (McCormick “Mucosal Inflammatory Response to Salmonella typhimurium Infection” Front. Immunol., 03 July 2014)
Gut Microbiota Species:
Gut microbiota of each warm blooded omnivorous animal, both avian and mammal, consists of about 1000 different bacterial speceis. Taxonomically, these speceis belong to two major phyla, Firmicutes and Bacteroidetes, followed by two minority phyla, Proteobacteria dn Actinobacteria, followed by phyla representatives that can be found only in some individuals. Each bacterial speceis present in gut microbiota has been subjected, as a metaorganism together with its host, to natural selection over millions of years of evolution. (Rychlik, “Ecological adaptations of gut microbiota members and their consequences for use as a new generation of probiotics” International J. Molecular Sciences, 2021).
The most frequently used probiotics belong mainly to genera Lactobacillus and Bifidobacterium. These genera are characterized by the production of organic acids that decrease pH and thus suppress the growth of competing microbiota. Lactobacilli are common in milk-fermented products, which are beneficial for animal or human hosts. Lactobacilli also commonly produce antimicrobial peptides, which may inhibit the browth of competing microiota. Different Lactobacillus species and their metabolic products exhibit immunomodulatory activites on vertebrate hosts, and supplementation of Bifidobacterium and Enterococcus probiotics decreased allergic rhinitis symptoms in children. (Rychlik, “Ecological adaptations of gut microbiota members and their consequences for use as a new generation of probiotics” International J. Molecular Sciences, 2021).
Lactobacilli: have been sued as probiotics for centruires; however, sequencing data shwo taht although Lactobacilli belong among common gut microbiota members in the small intestine, Lactobacilli do not dominate in the microbiota of distal parts of the digestive tract. Dominance of Lactobacilli in the small intestine, sometimes around 90% of all duodenal and jejunal microbiota, is due to their acid resistance, aerotolerance and rpaid multiplicaiton using energy from carbohydrate fermentation. Lactobacilli may degrade ogligosaccharides taht are difficult fro their host to digest into easily metabolized organic acids. They may also digest other substrates such as gluten and gliadin. Metabolic byproducts of Lactobacilli may act positively on human or animal perfrmance. The most relevant probiotic property of Lactobacilli is the rapid decrease in pH. A pH below 5 inactivates the majority of competing bacteria and makes such asn enviornment microbiologically safe for the human, pig or chicken host, cotaining non-thagoenic latic acid bacteria only. (Rychlik, “Ecological adaptations of gut microbiota members and their consequences for use as a new generation of probiotics” International J. Molecular Sciences, 2021).
Bifidobacterium: is anotehr genus known as a probiotic. It is phylogenitcally distant from Lactobacilli. They ahve a small genome size like Lactobacilli, but unlike Lactobacilli, the genome of Bifidobacteria is cahracterized by a high GC content of about 63%. Bifidobacteria exhibit a high level of resistance to different stress factors, which allows them to survive in an aerobic environment. (Rychlik, “Ecological adaptations of gut microbiota members and their consequences for use as a new generation of probiotics” International J. Molecular Sciences, 2021).
Protection of Mucosal Surfaces
Protection of mucosal surfaces against conolinization and invasion by microbes is provided by a combination of constitutive, non-specific substances (e.g., mucus, lysozyme, lactoferrin and definsins) and also by specific immune recognition that relies on secretory IgA (SIgA). In the GI tract, microbe sensing and processing occurs in the Peyr’s patch tissue consisting of mucosal epithelium and associated lymphoid tissue. Microbes are sabled and ingestedc by the M Cell which passes them to APCs. (Corthesy, “Recombinant immunoglobulin A:powerful tools for fundamental and applied research” Trends in Biotechnology, 20(2), 2002, 65-71
Consequences of Disturbing Gut Microflora
Antibiotic-associated diarrhaea (AAD): Diarrhae is a frequent adverse effect of antiotic treatment and a common condition in hospitalized patients. The mechasms by which antibiotics lead to AAD are multifaceted and include alteration of the normal intestinal microflora, often accompanied by overgrowth of patheogenic micro organisms. Nosocomial diarrhaea (NCD) is an entity defined as diarrhaea eveloped within 72 hf of hospitalisation (the 3 day rule). (Gorkiewicz, International J Antimicrobial Agents, 33(S1), 2009, S37-S41).
Gut Microbiome Influence on Obesity/Feeding:
Nutrients and microorganisms are among the most salient stimuli that the gut encounters. In the small intestine, epithelia neuropod cells rapidly detect nutrients and relay the sensory information, through the vagus nerve, to influence an anima’s appetite choices in real time. Evidence suggests that gut microorganisms, which are most abundant in the colon, substantially modulate feeding behavior, potentially through neuromodulators, immune signals and vagal pathway.
In the colon, vagal neurons from neuroepithelial circuits with neuropod cells, labelled by the neuromodulator PYY. These, and other colonic epithelial cells, are constantly exposed to microorganisms, which can be recognized by molecule patters such as flagellin, coollectively known as microbe assocaited molecular patters. Deleting TLR5 in all intestinal epithelial cells of mice leads to obesity, metabolic inflammation and spontaneous colitis.
In the gut, sensory epithelial cells are dispersed among other epithelial cells at a rate of less than 1 in 1k. They encompass two main lineages: those that express serotonin and substance P, and those that express several neuromodulators including cholescystokinin (CCK), glucagon-liek peptide (GLP-1) and PYY. The expression of these neuropeptides is preceded by that of the transcription factor Neurodl.
Flagellin action on TLR5: When mice were fasted overnight to induce hunger and then administered either flagellin or a PBS control by means of enema, the flagellin enema significantly reduced food intake within 20 min in littermate control mice but had no effect on mice lacking TLR in PYY labelled cells. The study showed that the ubiquitous microbial pattern flagellin stimulates TLR which leads to PYY release onto NPY2R vagal nodose neurons to regulate feeding. Mice lacking TLR5 in these cells eat more and gain more weight than control. The flagellin does not act on the nerve directly. Instead, flagellin stimulates neuropod cells from the colonic lumen to reduce feeding through a gut brain sensory neural circuit. (Bohorquez, “A gut sense for a microbial pattern regualtes feeding”
Synthetic Biotics:
Snthetic biotics involves use of genetic engineering to create new biological parts. Synlogic for example first looks at underlying mechanisms or metabolic dysfunctions that cause a disease. This involves a review of the existing literature to identify and validate potential molecular targets. For example, it assed GI component and role of diet restriction when evaluating for example PKU and HCU as potential target indications. PKU is a rare genetic disease caused by the inability to metabolize or break down phenylalanine (Phe), an amino acid found in many foods, including vegetables, breads and creals. Over time, the toxic building of Phe can lead to severe neurological disorders and developmental delays in patients. HCU is caused by the loss of functional of the enzyme cystathione beta-synthase, which results in the toxic building of homocysteine and its metabolites in human blood and urine. Symptoms include life-threatening strokes caused by thromboembolism, lens dislocation, skeletal abnormalities and intellectual disability. Similar to that for PKI, HUC disease management involves adopting a highly restrictive diet (low in methionine, a precursor to homocysteine). Using molecular biology techniques it next tests prototypes that can carry out specific functions such as consuming toxins, regulating cellular function and targetting drivers of disease. Then standard molecular techniques to construct bacterial strains with key elements for devlopment such as the generation of auxotrophs, which are unable to synthesize one or more essential growth factors. The synthetic biotics are grown under controlling manufacturing conditions, lyophilized and formulated into a stable powder that can be adminsitered orally. These syntehtic biotics are desigend to be nonreplicating, noncolonizing and reversible by rapid GI clearance. Synlogic has made progress in development of the investigational synthetic biotics SYNB1618 and SYNB1934, which are engineered strains of E. coli Nissle encoding phenylaanine ammonial lyase (PAS), an enzyme that breaks down Phe. They are designed to metabolize Phe in the GI tract and convert it into a harmless metabolite. Synlogic is also developing the SYN81353 synthetic bioc for the potential treatment of HCU. It is an engineered strain of E. coli Nissie that consumes methionine in the GI tract ot prevent the accumulation of hemocysteine. That strain was developed as part of a research collaboration with Ginkgo Bioworks.