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Engineered flora disorder sensing probiotics for clostridium difficile infection and recurrent infection management

A probiotics and expression cassette technology, applied in the field of engineered dysbacteriosis sensing probiotics for management of Clostridium difficile infection and recurrent infection, can solve problems such as unidentified specific species, unknown, etc.

Pending Publication Date: 2022-04-01
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, this strategy is a form of black-box engineering that does not identify the specific species of the microbiome required to suppress infection
Aside from what is believed to be a substantial replacement of the disrupted microbiome, the mechanisms behind the improved prognosis are largely unknown

Method used

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  • Engineered flora disorder sensing probiotics for clostridium difficile infection and recurrent infection management
  • Engineered flora disorder sensing probiotics for clostridium difficile infection and recurrent infection management
  • Engineered flora disorder sensing probiotics for clostridium difficile infection and recurrent infection management

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Engineering of a probiotic chassis without antibiotic selection

[0074] One of the challenges in implementing engineered probiotics in vivo is ensuring the long-term stability of the genetic circuits in the chassis. Engineered genetic circuits are often engineered on plasmids and maintained by antibiotic selection, but such techniques are not feasible for in vivo applications. For colonizing probiotics in the gastrointestinal tract, maintenance of plasmid stability by continuous antibiotic selection cannot be conveniently achieved.

[0075] To achieve a plasmid chassis system that does not require antibiotic maintenance, an auxotrophic phenotype was generated in the E. coli Nissle wild-type strain. The alanine racemase gene essential for D-alanine biosynthesis was deleted from the E. coli Nissle genome to generate strain EcN( Figure 5 A). D-alanine is required for bacterial cell wall biosynthesis. Deletion of D-alanine prevents further division of the bacteria, re...

Embodiment 2

[0079] Optimization of NanR-dependent sialic acid-inducible promoters

[0080] The sequence of the sialic acid-inducible promoter pNanA (SEQ ID NO:4) from the MG1655 E. coli genome was obtained from the EcoCyc database [Keseler, I.M. et al. Nuc.Acids.Res.41:605-12 (2013)]. The promoter Subcloned upstream of the gfp gene in the pEaat plasmid (pEaat-pNanA-gfp), and then transformed into T10 and EcN for expression characterization ( Image 6 A). Unexpectedly, the two E. coli strains showed different responses to sialic acid induction ( Image 6 B). Induction of sialic acid results in positive activation of the T10 pNanA promoter, resulting in GFP expression. On the other hand, GFP under the pNanA promoter in EcN was strongly expressed in the absence of sialic acid, but repressed when induced by high concentrations of sialic acid.

[0081] The difference in response indicates that pNanA is regulated differently between T10 and EcN. NanR (SEQ ID NO:5) was previously identified...

Embodiment 3

[0086] Characterization of engineered sialic acid biosensors under gastrointestinal-specific conditions

[0087]The parental strain of EcN preferentially colonizes the lower gastrointestinal tract, especially the colon and rectum [Sonnenborn, U. and Schulze, J. Micob. Ecol. Health Dis. 21:122-58 (2009); Schultz, M. Inflamm Bowel Dis. 14(7):1012-8(2008)]. This makes it ideal as a biosensor for dysbiosis in the lower gastrointestinal tract. Selected constructs were characterized under a range of conditions specific to the lower gastrointestinal tract to assess their suitability.

[0088] Note that the pNanA sequence contains a catabolite-activating protein (CAP) binding site at position 4→8. CAP is a transcriptional activator protein that initiates the transcription process when bound to circulating AMP (cAMP). cAMP levels are elevated in the absence of glucose, thereby effectively acting as a transcriptional activator in response to low glucose. EcNs containing inducible (p...

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Abstract

The present invention relates to methods of metabolic engineering of bacterial cells to produce bile salt hydrolases to inhibit germination of clostridium difficile endophytic spores and colonization within the human gastrointestinal tract. The bile salt hydrolase is operably linked to a sialic acid inducible promoter pNanA, wherein the pNanA is in turn controlled by a repressor nanoR. A recombinant bacterium expressing the bile salt hydrolase may be a probiotic strain to be used in the prevention or treatment of Clostridium difficile infection.

Description

technical field [0001] The present invention relates to methods of metabolically engineering cells to produce bile salt hydrolyzing enzymes to inhibit the germination and colonization of C. difficile endospores in the human gastrointestinal tract, probiotics and the prevention or treatment of C. difficile method of bacterial infection. Background technique [0002] Clostridium difficile (also classified as Clostridioides difficile) is a pathogen that can cause Clostridium difficile infection (CDI) and recurrent CDI (rCDI). CDI is one of the most common hospital-acquired infections worldwide. However, treatment of CDI is difficult due to bacterial endospore formation that evades antibiotic treatment. Recurrence of CDI occurred in 20.9% of patients and mortality due to these infections was 9.3%. It has been hypothesized that germination of dormant endospores after native microbiome disruption or dysbiosis leads to infection and relapse ( figure 1 ). Germination of endospo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55A61K38/50A61K35/741A61P31/04
CPCA61P31/04A61K35/741C12N9/80C12Y305/01024C12N15/70C12N15/74C12N15/635A61K38/00C12N9/88C07K14/245C12N1/20A61K35/74C12N1/205C12R2001/19A61K35/742A61K35/747A61K45/06C12N15/111C12N15/75
Inventor 许炜诠黄仁暎张旭
Owner NAT UNIV OF SINGAPORE
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