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Bifidobacteria as a model system for host-microbe interaction

Periodic Reporting for period 1 - BIF-PROTECTS (Bifidobacteria as a model system for host-microbe interaction)

Reporting period: 2021-04-01 to 2023-03-31

Our bodies are home to a staggering collection of microbes with, in particular, the human gut containing up to 2 kilograms of microbial mass. In healthy individuals the interplay between the immune system and the microbiota prevents tissue-damaging inflammatory responses towards commensal bacterial species, while permitting immune responses against infectious microbes. Changes in the gut microbiome have been linked to perturbation of immune function and several gut disorders such as inflammatory bowel disease (IBD), irritable bowel syndrome, and necrotising enterocolitis (NEC). Conversely, commensal microorganisms with purported health-promoting effects (probiotics) have been heralded as potential therapeutics for several ailments, the importance of which is illustrated by the currently 1,341 registered clinical trials using probiotics (based on the ‘clinicaltrials.gov’ database). Bifidobacteria, a group of Gram-positive commensals account for 475 of these trials. Bifidobacteria colonise the gastrointestinal tract immediately after birth, affect maturation of the host’s immune system during the neonatal period and can locally promote maturation of dendritic cells (DCs) in the gut. Lower counts of bifidobacteria have been observed prior to NEC diagnosis, suggesting a protective effect of this human gut coloniser in the context of this devastating disease, which occurs in 1 in 1000 live births and leads to death in 15% of pre-term infant patients. Interestingly, a meta-analysis of 20 clinical trials involving 5,529 infants found that prophylactic use of probiotic preparations containing Lactobacillus or Bifidobacterium significantly reduced the incidence of severe NEC and mortality in pre-term infants demonstrating a protective effect of these bacteria. Additionally, in pre-clinical mouse models, certain bifidobacterial strains can protect against diet-induced colonic mucus deterioration, diet-induced colitis, and intestinal immunopathology in the context of immune checkpoint blockade therapy. Despite the impact of microbes on our health and the large number of ongoing clinical trials, our current knowledge on how the microbiota, including probiotics, affects human health is based almost exclusively on descriptive and correlative studies with a pressing need for mechanistic studies. The evidence for the efficacy of probiotics in treating disease is often conflicting and confusing. This disconnect demonstrates that a thorough investigation of the underlying mechanisms beneficial to the host are needed to better translate research findings to the clinic and develop novel immunomodulatory therapies for infectious and chronic inflammatory disease with a known mode-of-action. The goal of BIFPROTECTS is to identify the molecular mechanisms by which Bifidobacterium breve induces a host protective state in innate immune cells and to phenotype commensal microbes for a similar effect. This proposal will use B.breve UCC2003 as a model system to identify underlying mechanisms through which this strain induces immunomodulatory host protective effects.
To identify microbial genes essential for host cell reprogramming a CRISPR interference system (CRISPRi) was developed to inhibit the expression of target genes via binding of the CRISPRi complex to the DNA target site in a sequence specific manner resulting in gene silencing. Inducible expression systems to control CRISPRi activity were similarly designed to allow for reversible control of gene expression. Several genes were targeted to validate gene silencing via CRISPRi, and the dCas9 system from Streptococcus thermophilus was able to repress target gene expression in a gRNA specific manner in the commensal bacteria Bifidobacterium breve UCC2003. Dual targeting of two genes simultaneously led to gene repression with similar efficacy as single gene targeting. However, when 4 genes were targeted simultaneously, the level of gene repression per gene is slightly diminished indicating a limit of how many genes can be targeted at once.
Continuous expression of the CRISPRi system can lead to escape mutants within the target population resulting in loss of target gene repression. Inducible expression of CRISPRi such that it is in an inactive state until required would prevent the development of escape mutants and increase the utility of the system. Existing systems to induce gene expression were found to be active in Bifidobacteria, however, high basal levels of dCas9 expression were observed indicating inadequate repression of the CRISPRi system demonstrating an inherent "leakiness" in these systems. further optimisation is required to ensure tight regulation of gene expression. An inducible nanoluciferase reporter system was developed to allow for rapid testing and optimisation of inducible gene expression systems in Bifidobacteria. This system could also be used to test different promoter constructs, response elements, and transcription factor binding sites in Bifidobacteria.
The current state of the art is to disrupt a gene by integrating a selectable marker via homologous recombination. This process is very inefficient occurring in less than 1 in 1 million transformed cells and is therefore restricted to a few strains of bacteria than are amenable to genetic manipulation. In contrast, the CRISPRi system developed during this project is active in every transformed cell, can be reversibly controlled by small molecules, and can be scaled up to interrogate gene function at a whole genome level. This will have a major impact on identifying the functions of genes in commensal gut bacteria and aid in the discovery of genes and pathways underlying the health benefits of probiotics.
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