(W1338) IN SILICO AND IN VITRO IDENTIFICATION OF ANTI-MICROBIAL PEPTIDES IN SYNERGY WITH MESENCHYMAL STROMAL CELLS AGAINST UROPATHOGENIC ESCHERICHIA COLI (UPEC).
Ph.D. Scholar Postgraduate Institute of Medical Education and Research Chandigarh, Chandigarh, India
Abstract: The increasing prevalence of infectious diseases and the limited effectiveness of conventional therapies highlight the urgent need for novel approaches to combat bacterial infections, including urosepsis. Prolonged antibiotic use fosters antimicrobial resistance, posing a global health threat. Mesenchymal stem/stromal cells (MSCs) offer promising therapeutic potential due to their regenerative, immunomodulatory, anti-inflammatory, and antimicrobial properties by secreting antimicrobial peptides (AMPs). This study explored the synergistic effect of MSCs and AMPs in combating pathigenesis by UPEC. We shortlisted six AMPs for in silico analysis targeting HisC, a key virulence factor of CFT073, followed by in vitro validation. The AMPs were docked with the HisC receptor (PDB ID: 1fg7) using the HDOCK web server. Molecular dynamics (MD) simulations were performed on the peptide-HisC complexes using gmx_qk software to evaluate key parameters: RMSD, RMSF, and binding free energy (BFE). The peptide with the lowest negative BFE was tested in vitro against CFT073 according to CLSI guidelines. A checkerboard analysis assessed the synergistic effect of the selected peptide and MSCs secretome. Biofilm formation and membrane disruption were evaluated by crystal violet staining and scanning electron microscopy (SEM). The Cecropin-LL-37 derivative (C-L) exhibited the lowest BFE (-1125.88 kJ/mol). MD simulation revealed RMSD and RMSF values of the Apomer and HisC-C-L complex (0.393 ± 0.086; 0.537 ± 0.354) and (0.140 ± 0.157; 0.197 ± 0.092). The antibacterial assay demonstrated a minimum inhibitory concentration (MIC) of C-L at 60 µg/mL. MSCs secretome also showed significant antibacterial activity against CFT073. Checkerboard analysis revealed that the combination both synergistically inhibited bacterial growth more effectively than each alone. This was further validated by significantly reduced biofilm formation and membrane disruption in the combination group. In conclusion, MSCs secretome and C-L peptide demonstrated significant antibacterial activity against CFT073 in vitro. Their synergistic effect will be further validated in a uroseptic preclinical mouse model.
