Behnsen et al. (2013). Probiotics properties, examples, and specific applications.

• Probiotics are beneficial microbiota components, used for centuries, known for their health impact on hosts.
• Probiotics modulate immunological, respiratory, and gastrointestinal functions.
• Escherichia coli Nissle 1917 and lactic acid bacteria are used to prevent/treat intestinal maladies like inflammatory bowel disease, constipation, and colon cancer.
• Engineering natural probiotics could further benefit the host.

Definition and History

• Probiotics are living bacteria that, when administered adequately, provide a health benefit.
• Eli Metchnikoff, considered the grandfather of modern probiotics, first attributed beneficial properties to some bacteria.
• Metchnikoff discovered in the early 20th century that "healthy bacteria," especially lactic acid bacteria (LAB), can positively influence digestion and the immune system.
• Most microorganisms recognized as probiotics are Gram-positive, with Lactobacillus and Bifidobacterium used for intestinal dysfunction treatments.
• The Gram-negative Escherichia coli Nissle 1917 (EcN), or "Mutaflor," is used in Germany to treat chronic constipation and colitis.
• Probiotics modulate immunological, respiratory, and gastrointestinal functions.
• Probiotics protect by competing with intestinal pathogens by releasing antibacterial substances like bacteriocins or metabolites.
• Advancements in probiotics arise from research on interactions between commensal microorganisms, pathogens, and hosts.
• Understanding gut colonization is essential for designing probiotics for specific uses.

Human Digestive Tract and Microbiota

• The human digestive tract has ecological niches populated by bacterial species with symbiotic relationships.
• The intestinal microbiota aids gut immune system development, food digestion, short-chain fatty acid and vitamin production and resistance to pathogenic microorganisms.
• The human gut microbiome consists of different bacteria species, some not well-known or characterized.
• The complexity of microbiota has recently been appreciated through deep sequencing, genomics, and metagenomics.

Distribution of Intestinal Microbiota

• The stomach is populated by <10² colony-forming units (cfu)/ml, including lactobacilli and streptococci.
• The Ileum and distal ileum are populated by 102–103 cfu/mL of bacteria, like E. coli, Klebsiella, Enterococcus, and Bacteroides.
• The Large intestine has the largest microbial population with 1010-1012 cfu/mL.
• Individuals have a specific "bacterial fingerprint" influenced by factors like maternal environment, host genotype, diet, and antibiotic treatment.
• The microbiota clusters in three enterotypes enriched in Bacteroides, Prevotella, or Ruminococcus.
  • Bacteroides uses carbohydrates for energy.
  • Prevotella uses mucins for energy.
  • Ruminococcus uses mucins and sugars for energy.
• Enterotypes are associated with long-term diets.
• Firmicutes and Bacteroidetes are the most common intestinal phyla, representing >90% of the microbiota, followed by Actinobacteria and Proteobacteria across all vertebrates.
• Bacteroidetes is represented by species including Bacteroides fragilis, with immunomodulatory capabilities thru its polysaccharide capsule
• Firmicutes is represented by class Clostridia, known to metabolize fiber and produce butyrate, a short-chain fatty acid with immunomodulatory activity.
• Class Bacilli bacteria, including Lactobacillus acidophilus and Enterococcus faecalis, makes up the rest of the Firmicutes phylum.
• Germ-free mice studies show that they have a thin intestinal epithelium, loss of short-chain fatty acid production, and immune system alterations.
• Microbiota administration restores functionality of the gut.

Microbiota Components

• Some microbiota components induce T-cell subsets development and defensins release.
• Normal individuals establish tolerance to commensal bacteria while maintaining an immune response against pathogens.
• Disruption of tolerance and immunity balance leads to intestinal pathologies including inflammatory bowel disease (IBD) and intestinal cancer.
• Immune response components, which orchestrate the mucosal barrier to microbiota, can contribute to IBD development if altered.
• An inflammatory response against the microbiota can change the host environment and affect the microbiota, exacerbating intestinal inflammation.
• Intestinal inflammation results in alterations to the microbiota.
  • Loss of Bacteroidetes and Clostridiales species.
  • Enhanced growth of Enterobacteriaceae.
• Antibiotic treatment is not recommended for self-limiting gastrointestinal infections due to alterations to the microbiota that can cause antibiotic-associated colitis.
• Restoring or augmenting the microbiota balance can provide resolution for a variety of diseases.
• Probiotics can be engineered to better mitigate particular intestinal pathologies by cytokine and enzyme delivery or competition with pathogens.

Most Characterized Probiotics

• Many culturable components of the microbiota are used for beneficial functions.
• Lactic acid bacteria (LAB) is a commonly used probiotic strains and are Gram-positive microbes used for centuries in food production (yogurt, cheese, pickles).
• Members of the LAB like Lactococcus and Streptococcus make up the endogenous microbiota in the human ileum and jejunum.
• The approval for LAB use in food, the absence of lipopolysaccharides (LPS), and lack of secreted proteases make many LAB ideal as probiotics.
• Recombinant proteins can be secreted without getting trapped in the periplasm.
• Lactococcus lactis is the most widely used LAB species for cytokine delivery.

Lactococcus lactis

• Unlike other Lactococcus species, Lactococcus lactis is only transiently present in the human gut and classified as noncommensal.
• Transient colonization of the probiotic strain assists protein/DNA vaccine delivery.
• The possibility to deliver proteins to the mucosa opens new therapeutic treatment methods in which traditional routes fail.
• The most commonly used Gram-negative probiotic strain is Escherichia coli Nissle 1917 (EcN).
• In 1917, army surgeon Alfred Nissle isolated during the First World War this strain from a soldier's feces who did not develop infectious diarrhea during a Shigella outbreak.
• Nissle's observation shows that EcN might provide mucosal pathogen colonization resistance.
• The probiotic effect and biosafety of EcN has been shown in trials and underlined by its long use in Central Europe.
• EcN is contained in Mutaflor and it's used for infectious diarrheal diseases and IBD treatment.
• Administering EcN to neonates prevents colonization of their digestive tract by multidrug-resistant pathogens.

Delivering Molecules to Reconstitute Barrier Defects Through LAB

• Recent probiotics research shows the use of LAB and delivering cytokines to target sites within a host.
• LAB-delivered cytokines can treat diseases that weaken the mucosal barrier (IBD subtypes Crohn's disease and ulcerative colitis).
• IBD is a Western world health concern, and a chronic inflammation of the intestine results in diarrhea, abdominal pain, and weight loss.
• IBD's etiology is unknown, but genetic alterations in host pattern recognition receptors and inflammatory genes and the microbiota contribute to the intestinal inflammation seen in IBD patients
• Treatments reducing intestinal inflammation are important.
• Interleukin 10 (IL-10) administration helps reduce chronic inflammation in IBD patients.
• IL-10 is a central role that down-regulates inflammatory cascades and establishes tolerance in the mucosa
• Human Clinical trials of subcutaneous administration of IL-10 faced several failures since direct oral administration not an option
• Schotte et al. (2000) engineered an IL-10-producing L. lactis strain and protects IL-10 degradation.
• Murine colitis improved following treatment with this IL-10-producing strain. onset of colitis was inhibited in IL-10-deficient mice, The amount of IL-10 needed for the observed reduction in colitis was several orders of magnitudes lower than systemic administration
• Delivery to the mucosa via LAB was key in the effectiveness of an IL-10-based treatment.
• Genetically modified organisms need biological containment for clinical probiotic applications.
• In L. lactis, the thymidylate synthase gene (thyA) replaced the human IL-10 gene.
• Thymidine or thymine is required in the media for thyA mutants to replicate, restricting strain growth to the human body and environment accumulation.
• The IL-10-producing L. lactis strain has been administered to 10 patients with Crohn's disease during a phase 1 clinical trial.
• Eight of 10 patients had a clinical benefit and five had complete clinical remission.
• These findings paved the way for subsequent trials.
• Strategies have been to induce cytokine secretion in the gut mucosa.
• Pathogenic yersiniae produce an anti-inflammatory protein called LcrV, which mediates immune response evasion of the host immune thru IL-10 production.
• An L. Lactis producing LcrV was able to reduce inflammation as efficiently as a L. Lactis strain secreting IL-10.
• Systemic administration of anti-TNF-a antibodies has drawbacks and limitations similar to systemic IL-10 administration.
• Production of the orally administered antibody and local administration at the side effects are low, which is helpful for inflammation of the gut.
• L. lactis produce anti-TNF-a antibodies as well as restores normal mucosal function
• Daily administration of L. lactis producing these nanobodies drastically reduced colonic inflammation.
• Therefore, LAB-mediated delivery of TNF-a and Anti IL -10 antibodies is more beneficial. - Heme oxygenase 1 (HO-1) improves integrity of the gut mucosal barrier.
• L. Lactis strains help to secrete the gut mucosa enzymes while decreasing the endotoxcemia.
• Heme oxygenase 1 (HO-1) reduces mortalities on mucosal injury.
• Another molecules that have a broad protective effect on the mucosa are peptides of the trefoil factor family (TFF).

TFF

• Human TFF-1 and -2 are produced by mucus-producing cells in the stomach and duodenum.
• TFF- 3 is highly expressed in goblet cells in the small and large intestine.
• As cytoprotective TFFs promote epithelial wound healing and reconstitution of the gastrointestinal tract, they become excellent candidates for mucosa restoration.
• These agents dont reach colon when administered orally.
• As TFFs bind to mucus and are absorbed in the cecum, intrarectal administration has proven effective.
• Intragastric administration of TFF-secreting L. Lactis to Mice works best for chronic and acute colitis.
• As TFF-1 and -3 are TFF-3 are located byhuman salivary glands in salivia, an L. lactis strain was recently formulated into a treatment.
• In cancer patients radio or chemotherapy, TFF works as an anticancer and affects mucosal cells leading to atrophy,swelling and ulceration.
• Probiotics are formulated in the mouth watch, and their L. Lactis strains are highly efficient in the healing.
• These studies underline the potential of using probiotics molecules directly at the targer in order to restore mucosal barrier function without interfering with systemic immunity.

E. Coli Nissle 1917 as a Probiotic

• It engineering Gram positive probiotic strains has been a uncomplicated as Gram negative probiotic strains is also useful.
• E. Coli secretes molecules such as molecules . - ecN block the infections - molecules like Peptide
• The secreted peptide has the ability to inhibit HIV fusion. The engineered the colonized and helps prevent HIV.
• When persistent Colonization IS Needed, EcN can be used unlike LAB. EcN delivers the epidermal growth factor
• EcN colonizes _EcN Enhane The wound healing.
• To enhance the efficacy and devlop probiotics, the ability of ecn colinizes must. -
• Ecn has shortened the duration of diaherria for pediatric children.
• Ecn Alleviates Intestinal Inflammation for patients with IBD & Ulcerative Colitis.
• Ecn Molecular helps Alleviate inflamtory disorders through
• ecn Administriation
• Helps againist protective fungal & bactrial including fungi.
• E coli inhibits The Inflammation.
• E colon enhances for mediated response thru pro-inflammatory locat cytokines.

ECN LIMITS INFLAMMATION

• The immune modulation effects have been observed through epithelial cells
• Ecn Enhances to improve colitis as its known
• Ecn modulates multiple was that balances E.COLI.
• The active of mmune response can affect immune systems.

E. Coli Activation Immune Types

• Many Differing genetic elements are found in different genetic components when bacterial strains in certain envoiroments
• Probiotics role in the adpation in bacteria.
• The strain is non pathogenic due the nature to serotype Lacks vilurnce

E. Col Feature In genome

• Important differnce between the sturcute differences
• Mutation
• This is a unique charcaterisitx
• The capsule stimulate tlr5 AND induce
• Iron multiple mechansms presences to multiple nuteirtent
• One uptake to to

Overall ecn

• Ecn maintance
• Ecn maintained
• This allows multiple in the bladder .
• Promotes the resident and promote the niche particualy is inflammed.
• Antimicrobials can be the for in addition to this ecisits protien Ecn shows signdincant induce in small intestine.
• High affinity transporters