Modified E. coli Nissle 1917 for Safe Probiotic Therapy
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Solution Overview
Problem
The ambivalence between the pathogenic and probiotic potential of Escherichia coli strain Nissle 1917 (EcN) poses a public health concern due to its production of genotoxins like colibactin, which is a virulence factor and potential procarcinogenic compound, necessitating the decoupling of genotoxic from probiotic activities.
Innovation Solution
By targeting the peptidase domain of the ClbP protein, specifically mutating residues like S95, K98, or Y186, to create a non-genotoxic EcN strain that retains antibacterial activity through siderophore-microcin production, thereby separating the genotoxic and probiotic functions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the wild-type EcN strain is used for probiotic treatment, then antibacterial activity against pathogens is improved, but genotoxicity and potential procarcinogenic effects worsen due to colibactin production
Solution Approach 1:
The invention extracts and removes the harmful peptidase domain from the ClbP protein through targeted mutation (e.g., S95, K98, or Y186 residues), thereby eliminating colibactin production while preserving the beneficial siderophore-microcin production pathway. This selective removal resolves the contradiction by keeping the protective function while discarding the toxic function.
Solution Approach 2:
The ClbP protein is functionally segmented into two domains: the peptidase domain responsible for colibactin activation (harmful) and the transmembrane domain involved in siderophore-microcin production (beneficial). By mutating specific residues in the peptidase domain, the invention selectively inactivates only the harmful segment while maintaining the beneficial segment's function.
2Object-generated harmful factors
If the pks/clb island is inactivated to eliminate genotoxicity, then safety is improved, but probiotic activity and antibacterial effects worsen
Solution Approach 1:
Instead of globally inactivating the entire pks/clb island, the invention applies a localized modification approach by mutating only specific residues (S95, K98, or Y186) within the peptidase domain of ClbP. This localized intervention selectively eliminates genotoxicity while preserving the overall probiotic functions encoded by the rest of the island, including siderophore-microcin production.
Solution Approach 2:
The invention changes the functional parameter of the ClbP protein by introducing point mutations that specifically alter peptidase activity while maintaining structural integrity and other functional domains. This parameter change approach allows selective elimination of colibactin production while preserving siderophore-microcin synthesis.
3Object-generated harmful factors
If the entire ClbP protein is inactivated to stop colibactin production, then genotoxicity is reduced, but siderophore-microcin production and antibacterial activity worsen
Solution Approach 1:
The invention segments the ClbP protein's functional domains and selectively targets only the peptidase domain for inactivation through residue mutation. This segmentation strategy ensures that the transmembrane domain and other functional regions remain intact and operational, preserving siderophore-microcin production while eliminating colibactin activation.
Solution Approach 2:
The invention extracts and removes only the harmful peptidase function from ClbP through targeted mutation of specific residues, rather than removing the entire protein. This selective extraction maintains the protein's structural framework and other functional domains necessary for siderophore-microcin production.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified EcN strain effectively reduces pathogen colonization and virulence while maintaining antibacterial activity, providing a safer and more effective probiotic solution for gastrointestinal disorders.
Implementation Method 1
hydrolyzed by the periplasmic membrane-bound ClbP protein with a peptidase activity, which releases the active colibactin
Implementation Method 2
The siderophore moiety is recognized by the catecholate-siderophore receptors of the target bacterium. The siderophore-Mcc can therefore enter and kill the sensitive bacterium by a 'Trojan Horse' stratagem, by mimicking the iron-siderophore complexes.
Implementation Method 3
Thanks to an extensive list of siderophores (enterobactin, salmochelin, yersiniabactin, and aerobactin) and multiple siderophore receptors and iron transport systems, EcN reduces S. Typhimurium intestinal colonization by competing for iron
Data Source
AI summary
The invention relates to the field of modified Escherichia coli strain Nissle 1917 (EcN) and its use for treating gastro-intestinal disorders. The invention is based on the study of the mechanisms implicated in the probiotic properties of the Escherichia coli strain Nissle 1917 (EcN). This study has allowed the inventors to decouple the probiotic activity of EcN from its genotoxic activity by demonstrating that EcN ClbP protein, the enzyme that activates the genotoxin colibactin, is also required for the siderophore-microcins activity of probiotic EcN, but interestingly, not its enzymatic domain that cleaves precolibactin to form active colibactin. Furthermore, inventors demonstrate in an in vivo animal model infected by a bacterial pathogen that administration of an EcN modified strain with clbP gene encoding ClbP protein inactive for the peptidase domain, is non-genotoxic (do not produce colibactin) but keeps the bacterial antagonist activity, and reduces colonization and virulence of the pathogen by maintaining the siderophore-microcin production. Thus this study opens the way to safe use of EcN and accordingly the present invention provides an Escherichia coli strain Nissle 1917 (EcN) bacterium carrying a gene encoding ClbP protein which is inactive for the peptidase domain, and its use as a drug and more particularly for use in the treatment of gastro-intestinal disease.