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Anti-Virulence Activity of 3,3'-Diindolylmethane (DIM): A Bioactive Cruciferous Phytochemical with Accelerated Wound
Karina Golberg1, Victor Markus2, Bat-El Kagan1
1Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.
Abstract:
Antimicrobial resistance is among the top global health problems with antibacterial resistance currently representing the major threat both in terms of occurrence and complexity. One reason current treatments of bacterial diseases are ineffective is the occurrence of protective and resistant biofilm structures. Phytochemicals are currently being reviewed for newer anti-virulence agents. In the present study, we aimed to investigate the anti-virulence activity of 3,3'-diindolylmethane (DIM), a bioactive cruciferous phytochemical. Using a series of in vitro assays on major Gram-negative pathogens, including transcriptomic analysis, and in vivo porcine wound studies as well as in silico experiments, we show that DIM has anti-biofilm activity. Following DIM treatment, our findings show that biofilm formation of two of the most prioritized bacterial pathogens Acinetobacter baumannii and Pseudomonas aeruginosa was inhibited respectively by 65% and 70%. Combining the antibiotic tobramycin with DIM enabled a high inhibition (94%) of P. aeruginosa biofilm. A DIM-based formulation, evaluated for its wound-healing efficacy on P. aeruginosa-infected wounds, showed a reduction in its bacterial bioburden, and wound size. RNA-seq was used to evaluate the molecular mechanism underlying the bacterial response to DIM. The gene expression profile encompassed shifts in virulence and biofilm-associated genes. A network regulation analysis showed the downregulation of 14 virulence-associated super-regulators. Quantitative real-time PCR verified and supported the transcriptomic results. Molecular docking and interaction profiling indicate that DIM can be accommodated in the autoinducer- or DNA-binding pockets of the virulence regulators making multiple non-covalent interactions with the key residues that are involved in ligand binding. DIM treatment prevented biofilm formation and destroyed existing biofilm without affecting microbial death rates. This study provides evidence for bacterial virulence attenuation by DIM.
Insights
3,3'-diindolylmethane (DIM), a phytochemical, effectively inhibits bacterial biofilm formation in pathogens like Acinetobacter baumannii and Pseudomonas aeruginosa. DIM also aids wound healing by reducing bacterial load and wound size, offering a novel anti-virulence strategy.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmacology
Background:
- Antimicrobial resistance (AMR) is a critical global health challenge, exacerbated by bacterial biofilms that render treatments ineffective.
- Phytochemicals are being explored as novel anti-virulence agents to combat resistant bacterial infections.
- 3,3'-diindolylmethane (DIM) is a bioactive compound found in cruciferous vegetables with potential therapeutic applications.
Purpose of the Study:
- To investigate the anti-virulence and anti-biofilm activity of 3,3'-diindolylmethane (DIM) against major Gram-negative pathogens.
- To elucidate the molecular mechanisms underlying DIM's effect on bacterial virulence and biofilm formation.
- To evaluate the efficacy of a DIM-based formulation in a preclinical wound infection model.
Main Methods:
- In vitro anti-biofilm assays on Acinetobacter baumannii and Pseudomonas aeruginosa.
- Transcriptomic analysis (RNA-seq) to assess gene expression changes in response to DIM.
- In vivo porcine wound healing studies using a DIM-based formulation.
- In silico molecular docking and interaction profiling of DIM with virulence regulators.
Main Results:
- DIM significantly inhibited biofilm formation in A. baumannii (65%) and P. aeruginosa (70%).
- Combination therapy with DIM and tobramycin achieved 94% inhibition of P. aeruginosa biofilm.
- DIM treatment reduced bacterial bioburden and wound size in P. aeruginosa-infected porcine wounds.
- Transcriptomic analysis revealed downregulation of key virulence and biofilm-associated genes and super-regulators.
- Molecular docking indicated DIM interacts with the binding pockets of bacterial virulence regulators.
Conclusions:
- DIM exhibits potent anti-biofilm activity and virulence attenuation against Gram-negative pathogens.
- DIM represents a promising therapeutic candidate for combating biofilm-associated infections and promoting wound healing.
- DIM's mechanism involves modulating bacterial gene expression and interfering with virulence factor production without causing direct cell death.
