The skin, the human body’s largest organ, is a crucial barrier against pathogens and environmental factors and plays significant physiological roles, including heat regulation and immune surveillance. Over 3,000 conditions can affect the skin, impacting nearly a third of the world’s population and profoundly affecting patients’ quality of life. These range from life-threatening diseases to chronic and socially impairing conditions. Environmental factors, such as excessive UV exposure, also significantly influence skin health, leading to premature aging and skin cancer. Current treatments often focus on symptom control and can cause side effects. Emerging treatments targeting underlying immune components can be costly.
Engineered live biotherapeutic products (eLBPs) present a promising approach, combining the natural adaptation of human microbes with synthetic design to enhance microbial capabilities. Cutibacterium acnes (C. acnes), the most abundant skin commensal, is an ideal chassis for skin eLBP development due to its location, high abundance, relevance in healthy skin, colonization capabilities, and low turnover. This bacterium colonizes the pilosebaceous units, making it a prime target for dermal biotechnological applications. Nevot, Santos-Moreno, et al. developed a synthetic biology toolbox for engineering C. acnes as a chassis for live biotherapeutics.
Methods
This study optimized a plasmid for replication in C. acnes, developed genetic parts for controlling and programming C. acnes behavior, and validated CRISPR interference (CRISPRi) for endogenous gene function. Auxotrophic strains were generated using homologous recombination, and transcriptional sensors were engineered for environmental signal detection. Finally, an antioxidant-secreting C. acnes strain was created to reduce oxidative stress in a UV stress model using keratinocytes.
Key Points
•Development of a Toolbox for C. acnes Genetic Engineering: A genetic toolbox was created for the traditionally intractable C. acnes to promote the development of microbiome-based skin therapies. The study optimized a plasmid from Propionibacterium freudenreichii for replication in C. acnes. The scientists removed non-essential sequences and adapted the plasmid for modular cloning, standardizing genetic parts with common prefix and suffix sequences. The toolbox includes basic gene expression tools, biocontainment strategies, markerless genetic engineering, and dynamic transcriptional regulation.
•CRISPR Interference for Quick Prototyping of Auxotrophies for Biocontainment: CRISPRi was used to screen metabolic genes, potentially leading to single-gene knockout amino acid auxotrophies. The goal was to generate an auxotrophic strain without any heterologous antibiotic resistance gene. CRISPRi knockdowns impaired bacterial growth when the amino acid was not supplied. Lysine and proline biosynthesis pathway repression showed the highest growth defect compared with a dCas9-only control.
•Engineered Sensing and Actuation in C. acnes: Different transcriptional sensors were engineered in C. acnes following two main strategies: the transplantation of known transcription factors (TFs) from other organisms and the de novo discovery of natural genetic resources from C. acnes. Temperature-change detection has been previously used to trigger the self-destruction of bacteria leaving the body (i.e., kill switch) for biocontainment. The researchers exposed C. acnes to a 42°C heat shock for 15 min and looked for upregulated genes with RNA-seq.
•Antioxidant-Secreting C. acnes Reduces Oxidative Stress: An antioxidant-secreting C. acnes strain was engineered for reducing oxidative stress. Superoxide dismutases (SODs) neutralize the superoxide anion and protect cells from oxidative damage. The SodC-F1 from the enterohemorrhagic E. coli O157:H7 was coupled with a secretion signal from a highly secreted endogenous protein, RoxP14. The SodC-F1 produced by C. acnes was able to significantly reduce UV-mediated ROS levels in keratinocytes.
This research establishes C. acnes as a promising chassis for eLBP development, offering a versatile toolbox and antibiotic-free methodologies for safe skin applications. The ability to engineer C. acnes to detect artificial and environmental cues, along with the demonstration of oxidative stress reduction, paves the way for microbiome-based skin therapies. Making these tools available to the research community can accelerate skin microbiome research using the most abundant skin commensal.
Link to the study:
https://www.cell.com/cell-systems/pdfExtended/S2405-4712(25)00002-X
