Epidermal growth factor (EGF) was originally isolated from mouse salivary gland extract as a factor accelerating corneal wound healing (10). EGF now is recognized as a general growth factor that exerts various actions, including cell migration and proliferation, on a wide variety of cells (7, 43, 44).
EGF stimulates the proliferation of keratinocytes in culture, and the topical administration of EGF accelerates the epidermal regeneration of partial-thickness burns or split-thickness incisions in vivo. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein-tyrosine kinase activity of the receptor. Although EGFR activation by its ligands, including transforming growth factor- α (TGF- α ), heparin-bound EGF, betacellulin, and amphiregulin, can regulate many cellular processes (12), the main physiological roles of EGF are associated with epithelial wound healing.
Upon exposure to ulcerogenic and/or necrotizing agents, such as aspirin, indomethacin, bile acids, alcohol, and ischemia, the gastrointestinal mucosa develops characteristic morphological, ultrastructural, and functional changes reflecting the disruption of the mucosal barrier. The healing of deep mucosal erosions requires the reconstruction (reepithelialization) of the surface glandular epithelial structures and the lamina propria, including the mucosal microvascular network, nerves, and connective tissue cells. The gastrointestinal mucosa has a remarkable ability to repair damage with the support of EGF, which stimulates cell migration and increases blood flow (3, 5, 27, 36, 45).
During reepithelialization, the repair of deeper injuries (erosions) requires epithelial cells to compensate for the mucosal defect.
EGF, which is mitogenic for progenitor cell populations, increases the release of gastric mucin, attenuates gastric acid secretion, and stimulates cell migration (9).
Only a small fraction of orally administrated mucoactive biotherapeutics, including recombinant protein drugs, reaches the intended target site because of strong digestive degradation in the gastrointestinal tract. As a consequence of inefficient drug delivery, it is important to develop new methods for the localized delivery of mucoactive biotherapeutics.
The basic idea in the present study was derived from the assumption that intestinal commensal bacteria or probiotics can carry and deliver key medicinal components to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease.
Genetically modified probiotics might be a good alternative in mucosal biotherapy as a live safe carrier. Escherichia coli strain Nissle 1917 (O6:K5:H1) is a nonpathogenic fecal isolate that has been used as a probiotic agent in human and animal medicine since the early 1920s to treat chronic inflammatory and infectious diseases of the human and animal intestine (15, 16, 29).
Probiotics are defined as viable microorganisms with physiologically beneficial or therapeutic activities. Various in vitro and animal studies with probiotics, including E. coli Nissle 1917, have demonstrated the capacity of probiotics to reduce intestinal inflammation (16, 20), strengthen the integrity of the intestinal epithelial barrier (42, 47), lessen host hypersensitivity (4), or suppress epithelial adherence and invasion of pathogenic bacteria (31, 33).
Moreover, limited clinical investigations using E. coli Nissle 1917 and other microorganisms have demonstrated that probiotic-based therapeutic application can be efficacious in patients with chronic ulcerative colitis (13, 23, 32) and irritable bowel syndrome (38). E. coli Nissle 1917 is relatively safe for therapeutic applications, although its administration to immunocompromised hosts with defective intestinal microbiota after antibiotic therapy may lead to severe adverse effects (21). Regardless of the high numbers of E. coli Nissle 1917 cells that can colonize the intestinal tract, the bacterium does not cause colitis even in gnotobiotic animals monoinoculated with the strain (21).
Molecular genetics as well as functional analyses have revealed that E. coli Nissle 1917 does not produce any virulence factors or carry any genes for pathogenicity traits, and it does not form enterotoxins, cytotoxins, or hemolysins (6, 39). The collective observations support the general recognition of biotherapeutic applications using E. coli Nissle 1917 as a safe organism for human use. Type I secretion occurs in a continuous process across both the inner and the outer membrane of Gram-negative bacteria. One representative type I transporter is the ATP binding cassette (ABC) protein, which recognizes the C-terminal signal sequence of the target protein and hydrolyzes ATP for protein translocation (11, 28).
In this study, we focused on the potential of E. coli Nissle 1917 as the vehicle for the targeted delivery of therapeutic proteins to ulcerated intestinal epithelia. E. coli Nissle 1917 secreting human EGF in conjunction with the lipase ABC transporter recognition domain (LARD) by the ABC transporter system (8) was established. In advance of the clinical application of the recombinant probiotic, the secretory and delivery systems were biologically assessed in human intestinal epithelial monolayer cell cultures. In particular, the recombinant bacterially mediated wound-healing process in the ulcerated epithelial layer was quantified and was shown to be mechanistically associated with EGFlinked receptor signals. The present study provides a basis for the clinical application of human biotherapeutics with efficiency and enhanced safety via the probiotic E. coli Nissle 1917.