AUTHOR=Bennett Aliyah N. , Maziarz Jacob F. , Laipply Baileigh , Cole Allysa L. , Woolard Katherine J. , Sorge Amy , Zeiler Michael J. , Melander Roberta J. , Melander Christian , Gunn John S. 

TITLE=Mechanisms of antibiofilm compounds JG-1 and M4 across multiple species: alterations of protein interactions essential to biofilm formation

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 15 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1631575

DOI=10.3389/fcimb.2025.1631575

ISSN=2235-2988

ABSTRACT=The majority of human bacterial pathogens have the ability to form biofilms in vivo on body tissues and implantable medical devices. Biofilm-mediated chronic bacterial infections are difficult to treat due to their recalcitrance to antimicrobials and immune effectors, often requiring invasive surgical intervention to clear the infection. The difficulty in effectively executing these treatment strategies underscores the need for therapeutic agents that specifically target the biofilm state. To this end, we previously identified two small molecules, JG-1 and M4, that in vitro effectively inhibit and disperse biofilms of Salmonella Typhimurium and members of the ESKAPE pathogen group, including Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In addition to its antibiofilm effects, M4 has a bactericidal effect on Staphylococcus aureus and Enterococcus faecium. While these compounds have promising utility as antimicrobial agents, their mechanism of action remains unknown. By employing multiple techniques including RNAseq, thermal proteome profiling, and site directed mutagenesis, we identified multiple proteins essential to biofilm formation and evaluated their role in the presence of JG-1 and M4 in mutant and wildtype backgrounds. We report that the JG-1 and M4 actions are influenced by proteins important to biofilm maintenance, including OmpA, OmpC, and TrxA. Compound-bacteria interactions cause transcriptional changes that result in biofilm dispersal, and modulation of other virulence mechanisms, including invasion and motility. Additionally, we report that M4 interacts with S. aureus CodY, which promotes cell death, while the specific targets in S. Typhimurium and E. cloacae remain elusive. Collectively, this study presents an empirical investigation into JG-1 and M4’s mechanism of action in S. Typhimurium, E. cloacae, and S. aureus, and how the antibiofilm compounds disrupt microbial community dynamics, ultimately driving biofilm dispersal or cell death.