Advancing a cell-free protein synthesis system suitable for fundamental and applied research and assessing the antimicrobial efficacy of peptides targeting the ribosomes of ESKAPE pathogens

The rapid global rise of multidrug-resistant bacteria, particularly within the high-priority ESKAPE pathogen group (including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), poses a critical threat to modern medicine and demands urgent innovation in antibiotic discovery. To combat antimicrobial resistance, modern drug discovery must shift its target away from traditional broad-spectrum antibiotics and focus on species-specific, precision medicine. Although the bacterial ribosome remains a highly effective target for the development of selective therapeutic candidates, traditional screening approaches face significant bottlenecks as the cellular envelope hinders the promising molecules’ access to the translational machinery. The cell-free protein synthesis (CFPS) system not only eliminates this biological barrier, giving potential inhibitors direct access to the ribosomes, but also offers the capability to mimic the native pathological environment. This research study established a comprehensive CFPS pipeline that progresses both screening throughput and pathogen specificity. In project 1, a rapid, non-hazardous assay named FAST (Fluorescent Assembly of Split- GFP for Translation tests) was developed to efficiently monitor protein synthesis in vitro. The platform relies on the reassembly of a large, non-fluorescent fragment (GFP1-10) and a small peptide tag (GFP11), where GFP11 is fused to a protein of interest to monitor its active synthesis based on the fluorescence produced by complementation of the two fragments. The GFP1-10 was fused to Maltose Binding Protein (MBP), this MBP fusion prevents aggregation and improves solubility, while also acting as a strategic purification anchor. The platform’s capacity to monitor translation was successfully validated by tracking the synthesis of four distinct proteins spanning different amino acid lengths: CspA (70), HupB (90), MoaB (170) and NadE (275). Furthermore, the platform proved highly suitable for screening antimicrobial compounds targeting the translational machinery, using the aminoglycoside antibiotic Kanamycin as a positive control. To bridge the “relevance gap” between standard laboratory strains like Escherichia coli, and ESKAPE bacteria, project 2 expanded the CFPS system by establishing a species- specific screening platform using native extracts from high-priority pathogens. While E. coli is widely used, it can obscure the direct ribosomal susceptibility of specific molecular targets. This project successfully optimized a customized protocol to extract active 70S ribosomes from six ESKAPE strains. The native 70S ribosomes were utilized to assess the antimicrobial activity of two proline-rich antimicrobial peptides (PrAMPs): Bac7(1-16) and B7-005, which are variants of the mammalian peptide Bac7, which interferes with protein synthesis by binding to the ribosomal exit tunnel. The inhibition of these two AMPs was quantitatively measured using a firefly luciferase (Fluc) encoded by a reporter mRNA. This research provides original, comparative data detailing the inhibition capacity of these two peptides within a native, pathogen-species cell-free environment. Altogether, this work supports global efforts to address antimicrobial resistance by introducing molecular tools designed to meet the precision demands of modern antibiotic discovery.