Potential of Escherichia coli Probiotics for Improved Health and Disease Management

Although natural gut microbiota contains Escherichia coli as a commensal, this bacterium, along with other members of the Enterobacteriaceae family, are usu-ally known for their pathogenic potential. Interestingly, E. coli colonizes first and remains all through life, and in fact, some strains possess beneficial properties such as antibacterial colicin secretion. Among the beneficial strains, E. coli Nissle, isolated in 1917, has been the most extensively explored strain. Adaptability to survive under diverse conditions coupled with facile genetic manipulations enabled the design of E. coli strains with properties to deliver antioxidant, anti-inflammatory, and antitumor molecules. Moreover, genetically modified E. coli strains secreting enzymes for converting sucrose and fructose into insulin and mannitol, respectively, were very effective in preventing the onset of metabolic disease by acting as synbiotics. Thus, E. coli is emerging as a very potent probiotic platform for developing strains with the potential of controlling many metabolic and multifactorial diseases, including cancer.


Introduction
Escherichia coli resides in the gastrointestinal (GI) tract of animals along with a few hundreds and thousands of different microbiota. In humans, E. coli is present at less than 1% of gut microbiota and it is not among the 25 most prevalent bacteria [1,2]. But E. coli is the predominant Enterobacteriaceae species in humans [3]. Interestingly, E. coli is the first to colonize the intestines and persists all through life in humans [4]. Mucin layers do not allow any direct interaction of gut microbiota with the enterocytes. However, the diversity of gut microbiota is known to influence the intestinal permeability involving LPS, peptidoglycan, lipoproteins, deoxynucleic acid (DNA), and ribonucleic acid (RNA). Most E. coli strains are nonpathogenic and exist as commensals, but some pathogenic strains are associated with severe diseases [5]. Additionally, some E. coli strains known as pathobionts do not cause any disease in healthy individuals but exacerbate chronic inflammatory diseases [1]. The E. coli population in the GI tract is dynamic with a turnover in months to years [3,6]. Humans contain five different strains of E. coli [7]. Oxygen diffusion from intestinal epithelium is favorable for Enterobacteriaceae members including E. coli to be present in close proximity to the mucus layer [8]. E. coli is known to play an important role in the maintenance by decreasing oxygen content, has low levels of smooth lipopolysaccharide (sLPS) [23]. EcN prevents the colonization of pathogens by efficient adhesion with the help of fimbriae and capsule to the epitheliumt but not activating inflammation as its lipopolysaccharide (LPS) has a short O chain and weak binding to toll-like receptor 4. EcN decreases pro-inflammatory cytokine and increases anti-inflammatory cytokine formation [24]. EcN repairs leaky gut by increasing the expression and phosphorylation of tight junction protein zonula occludens-1 (ZO-1), ZO-2, and claudin 14 [25][26][27]. Additionally, EcN prevents disruption of epithelial tight junctions by inhibiting NF-κB-mediated activation of the MLCK-P-MLC signaling pathway [28]. EcN mediates pathogen elimination by secretion of low molecular weight microcin H47 and microcin S. Probiotic EcN, but not commensal E. coli MG1655, increases serotonin (5-hydroxytryptamine) secretion by enterochromaffin cells [29]. Interestingly, bacteria are known to secrete vesicles known as membrane vesicles (MVs) [30]. Gram-negative bacterial outer membrane vesicles contain LPS and the size and complexity of O-antigen, the number, and nature of fatty acid components of lipid A determining the beneficial or toxic effects on the host cells. EcN outer membrane vesicles (OMVs) prevent the inflammation and progression of dextran sodium sulfate (DSS)-induced colitis in mice [26,31,32]. EcN OMVs get internalized by macrophages and activate the phagocytosis, which increases pro-inflammatory cytokine secretion and killing of pathogens [33].

Symbioflor-2
Symbioflor-2 is a commercial product containing six E. coli strains, which brings about an increase in β-defensin-2 and reduces mast cell activation [19]. Symbioflor-2 is effective in reducing symptoms of irritable bowel syndrome [34]. Microcin S is produced by Symbioflor G3/10 strain. Surprisingly, virulence genes have also been detected in Symbioflor-2 genomes suggesting that the presence of virulence genes does not imply pathogenicity [2]. Transcriptomic analysis of ileum and colon upon inoculation with Symbioflor-2 strains indicated the increase in defense responses involving dual oxidase/nitric oxide pathway mediated reactive oxygen species generation along with β-defensin-2 activity [35]. Transcription profiles were distinct with EcN and Symbioflor-2.

Colinfant
Colinfant is an E. coli (A0 34/86) strain that is used as prophylactic in infants for allergy, nosocomial infection, and diarrhea [20,21]. Additionally, it is effective in later years in preventing infections and developing allergies. Some strains of Klebsiella oxytoca are implicated in antibiotic-associated diarrhea, which could be reduced by the administration of Colinfant in infants. Colinfant also prevents infection of pathogenic E. coli.

Genetic modifications of probiotic E. coli
Probiotic E. coli strains have been modified to improve colonization, to secrete metabolites, proteins, and enzymes exploiting a variety of genetic manipulations ( Table 2). EcN was tagged with a green fluorescent protein (Gfp), which facilitated monitoring the colonization and survival in stomach, ileum, colon, and Peyer's patches [36]. EcN was detected in the fecal matter at 45 days after oral inoculation. EcN contains two cryptic plasmids MUT1 and MUT2, and these plasmids were cured using CRISP-Cas9-assisted double-strand breaks [47]. EcN strain cured of these plasmids had similar growth under Luria broth conditions despite differences in the DNA content. Effects of colonization and survival of the plasmid-cured strain with decreased DNA content as compared to the wild-type strain need to be investigated to determine the impact of metabolic load. Alternatively, both the cryptic plasmids of EcN have been engineered for stable maintenance and expression of recombinant proteins [53].
Vitreoscilla hemoglobin (VHb) with a high affinity for oxygen facilitates the survival and functionality of bacteria under microaerobic conditions [54] promoted colonization of genetically modified E. coli in the gut. E. coli 16 double transformants of gfp and Vitreoscilla hemoglobin (vgb) genes at 10 8 cfu/g were present in the rat fecal matter after 70 days of oral administration, while Ec16 gfp was not found after 48 days [37]. Additionally, catalase activity of VHb scavenges the reactive oxygen species, which decreased the carbon tetrachloride-induced hepatotoxicity in rats.
Pyrroloquinoline quinone (PQQ ) is a water-soluble antioxidant with the highest redox cycles of 20,000, promotes mitochondrial biogenesis and cellular signaling, and provides health benefits [55]. E. coli 16 strain tagged with gfp-vgb genes and transformed with pqqABCDE operon from Pseudomonas fluorescens Bf1 prevented colon and liver damage by dimethylhydrazine (DMH) due to the combined beneficial effects of effective colonization and antioxidant properties of Vhb and PQQ, respectively [38]. DMH had systemic oxidative damage, and decreased brain serotonin and norepinephrine levels, but epinephrine levels were increased [39]. In addition to decreasing the oxidative damage, E. coli 16 vgb-pqq strain had near-normal levels of neurotransmitters in rats. These beneficial effects  were not similar with treatments of Ec16, vitamin C, or PQQ alone suggesting other than its additional ability to confer antioxidant properties, probiotic E. coli 16 had synergistic effects related to the continuous secretion of PQQ in the gastrointestinal tract. These beneficial effects were also seen in EcN strain that was modified in a similar manner to that of Ec16 strain [40]. EcN vgb-pqq recombinant strain effects were monitored in rats for alcohol toxicity in chronic and acute exposure. Chronic alcohol caused extensive oxidative damage and induced hyperlipidemia and the EcN::vgb-gfp(pqq) probiotic strain prevented the deleterious effects, while EcN, PQQ, and vitamin C alone had no significant effects. These effects were also correlated with increased short-chain fatty acids (SCFA) in the colon. However, oral PQQ had better effects than recombinant EcN strain in acute alcohol damage. These studies further supported the significance of endogenous PQQ biosynthesis by probiotic E. coli.
Aging is associated with progressive loss of tissue functions mediated by reactive oxygen species-induced oxidative damage as a result of mitochondrial dysfunction [56][57][58]. EcN::vgb-gfp transformed with pqq gene cluster from Gluconobacter suboxydans 621 decreased the rotenone-induced mitochondrial oxidative damage in aging rats along with decreased lipogenesis and increased fatty acid oxidation genes correlated with increased colonic SCFA and PQQ in both feces and liver [41]. Additionally, an increase in mitochondrial biogenesis and metabolism indicates delaying of age-related tissue damage.
Heavy metal toxicity is mediated by reactive oxygen species [59]. Chelation of heavy metal ions and antioxidants is used to prevent the toxicity. EcN::vgb-gfp strain operon containing pqq gene cluster from Gluconobacter oxydans decreased the Cd and Hg toxicity upon oral supplementation citric acid due to the antioxidant effects of PQQ and chelation ability of citric acid [42]. Subsequently, EcN::vgb-gfp strain containing pqq gene cluster from A. calcoaceticus and gluconate dehydrogenase (gad) operon from Pseudomonas putida KT2440 secreted PQQ, gluconic and 2-ketogluconic acids, and this strain prevented toxicity caused by Cd, Hg and Pb without affecting the essential metal ions [43]. Thus, 2-ketogluconic acid produced by EcN recombinant strain is mimicking the chelating abilities of citric acid. Similarly, EcN strain containing As(III) S-adenosylmethionine (SAM) methlyltransferase (arsM) and pqq gene cluster prevented arsenite toxicity by scavenging arsenite-induced reactive oxygen species by secreted PQQ and converting arsenite into non-toxic trimethylarsenite in rats [44].
EcN recombinant strain containing pqq operon secretes 15 mM gluconic acid [43]. Gluconic acid was proposed for cancer therapy as cancer cells utilize citrate for growth and gluconic acid irreversibly inhibits citrate transporter, which is expressed on cancer cells [60]. Hence, EcN producing gluconic acid could prevent the progression of tumors, especially colorectal cancers. Staphylococcus aureus α-hemolysin expressing EcN recombinant strain forms pores in the tumor cells resulting in the regression of tumors in mice [51]. Similarly, tumor regression also occurred in mice xenografted with human colorectal cancer cells treated with EcN strain expressing hemolysin E (HlyE) a pore-forming protein [52].
SCFA such as acetate, propionate, and butyrate produced by gut microbiome is necessary for the survival of colonocytes, maintenance of intestinal integrity, mucus production, serotonin release by enterochromaffin cells, and secretion of gut hormone peptide YY in the intestine [61,62]. Additionally, SCFA also regulates brain and liver functions while diminished SCFA signaling is associated with metabolic diseases [63]. Propionate and butyrate prevent the progression of these metabolic diseases [64]. In order to design EcN to secrete butyric acid, fumarate reductase (frdA), lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE), and phosphotransacetylase (pta) genes involved in the fermentation product Potential of Escherichia coli Probiotics for Improved Health and Disease Management DOI: http://dx.doi.org /10.5772/intechopen.100380 formation of succinic, acetic, and lactic acids were deleted to generate EcN YF005 strain [49]. The atoDABE operon encodes the genes for the formation of acetoacetyl CoA and butyryl CoA to the butyric acid formation, while hbd and crt from Clostridium acetobutylicum and ter from Treponema denticola genes convert acetoacetyl CoA into butyryl CoA. The native promoter of atoDABE operon was replaced with a strong, constitutive P L promoter from phage λ, and synthetic P L -LacO-hpdcrt-ter operon was integrated at methylglyoxal synthase (msgA) gene to generate EcN Y2023 strain. This strain produced 0.49 g/L butyric acid on glucose. It will be interesting to determine its therapeutic potential in animal studies.
EcN deletion mutant of dapA gene coding for 4-hydroxytetra-hydropicolinate synthase was generated for incorporating phenylalanine degradation for the treatment of phenylketonuria [48]. Two different SYNB1618 strains were generated by incorporating phenylalanine ammonia-lyase and L-amino acid deaminase (pma) genes, which convert phenylalanine into trans-cinnamic acid (TCA) and phenylpyruvate, respectively. In humans, TCA is further transformed into hippuric acid in the liver and excreted in the urine. The oral load of 70 mg phenylalanine was reduced by 58% in the serum samples of individuals fed with the modified strain.
EcN was genetically modified for inflammatory bowel disease by probioticassociated therapeutic curli hybrids (PATCH) approach using a fusion protein of amyloid domain for self-assembly (CsgA) linked to trefoil factor-3 with 6 His residues [50]. Oxidatively damaged inflamed regions are conducive for the growth of facultative anaerobes. Consequently, modified EcN strain numbers increased at the damaged regions and secreted curly fibers that facilitated the repair of damaged regions. The EcN-engineered strain could ameliorate the weight loss in DSSinduced colitis in mice.
EcN expressing a fusion protein of cholera toxin B domain and insulin growth factor-1 (CTB-IGF1) was proposed as a long-term therapeutic strategy for diabetes [65]. It was hypothesized that EcN expresses the fusion protein in the intestine that would cross the intestinal epithelium into blood circulation facilitated by CTBspecific interaction with GM1 ganglioside oligosaccharide and IGF will activate the insulin effects.

E. coli as synbiotic
The beneficial effects of probiotic E. coli strains are contributed by their functions in the small intestine as well as in the colon. However, prebiotics are nutrients for the survival and maintenance of the colonic microbiome, which secrete host-beneficial SCFA as fermentation products [66]. Synbiotics are a mixture of prebiotics and probiotics, which provide synergistic effects of both components [67]. Dietary fructose and sucrose are implicated in the onset and progression of metabolic diseases [68,69]. EcN::vgb-gfp was modified with two different synthetic operons containing Ptac-pqq-glf-mtlK and Ptac-pqq-fdh that convert dietary fructose into mannitol and 5-keto-D-fructose that are prebiotics in the small intestine [45]. These prebiotics then serve as nutrients for colonic bacteria to produce SCFA. PQQ secreted by the synbiotic EcN will scavenge reactive oxygen species produced by fructose. Both mannitol and 5-keto-D-fructose producing strains demonstrated synbiotic activities by preventing dietary fructose-mediated metabolic disorders in rats. Fructose is known to improve iron status by its reductive ability compared to other sugars [70]. However, metabolic complications of fructose hindered its applicability. Since EcN synbiotics overcome the negative effects of fructose, these strains were found to also improve iron status [71].
High dietary sucrose also contributes to the metabolic disorders [68]. Inulosucrase catalyzes the conversion of sucrose into inulin [72]. Probiotic Ec16 transformed with inulosucrase of Lactobacillus johnsonii NCC 533 resulted in its secretion in the supernatant, while the enzyme was localized in the periplasm of E. coli BL21 suggested that extracellular enzyme in Ec16 could get transported using colicin E1/1a1b transport system [22]. EcN genomic integrant with vgb-gfp-pqqABCDE-inuJ gene cluster prevented high sucrose-induced metabolic disorders in rats by increased PQQ and SCFA [46].

Conclusion
The potential of probiotic E. coli is increasing over the years starting from maintaining the intestinal barrier in healthy individuals to the treatment of complex diseases such as colorectal cancer and inflammatory bowel disease. Since many commercial E. coli products were found to deviate by orders of magnitude in terms of claimed numbers, monitoring strain identity and numbers is imperative for exploiting their complete potential [73]. Distinct properties of E. coli probiotics coupled with the ease of developing strains with desired traits could greatly expand their applications.