Development of Recombinant Antibiotics Against Enterococcus Faecium

Abstract

Purpose: It was aimed to develop a recombinant antibiotic originating from the VH sequence of antibodies obtained from PBP5-immunized mice Materials and Methods: The gene encoding soluble PBP5 was cloned into the pET-30a(+) vector, produced recombinantly, and purified by IMAC. CD-1 mice were immunized with sol-PBP5, and the immune response was confirmed by ELISA. RNA from immunized mice spleens was used for cDNA synthesis and VH amplification. A VH-displaying phage library was created by cloning VH amplicons into pComb3-hy phagemid. Phages binding to sol-PBP5 were selected via biopanning, and their VH sequences were identified. The 3D structure of VH43 and its interaction with sol-PBP5 were modeled, and VH43 was produced recombinantly. Binding tests were performed with BOCILLIN-FL. Synergistic effects of VH43 with cephalosporin were tested using bacterial growth analysis and modified Kirby-Bauer/spot tests. Results: Cloning of sol-PBP5 into the pET-30a(+) vector was confirmed by sequence analysis. Expression and purification were validated via SDS-PAGE, with BOCILLIN-FL treatment confirming the protein's native conformation. Immunization was confirmed by ELISA (p-value = 9.02e-05). cDNA from total RNA of immunized mice spleens showed 500-600 bp bands on agarose gel. VH amplicons were cloned into the phagemid, and a phage library was produced via M13K07 infection. Affinity phages for sol-PBP5 were selected by biopanning, and VH sequences were determined after the third biopanning cycle. VH43's amino acid sequence and CDRs were identified, with a molecular weight of 10.3 kDa, 12 alpha helices, and 61 beta chains. Seven hydrogen bonds were found between VH43 and sol-PBP5. Recombinant VH43, confirmed via SDS-PAGE to be 20.3 kDa including His-tags, showed reduced brightness in BOCILLIN FL binding experiments. Combining VH43 with cephalosporin significantly inhibited bacterial growth, with increased zone diameter in modified Kirby-Bauer tests compared to cephalosporin alone. Conclusion: The increase in antibiotic-resistant E. faecium strains requires the development of new molecules. VH43 discovered in this thesis study is the first antibody/antibody fragment developed against E. faecium and whose sequence was determined. Combining recombinant VH43 with cephalosporin showed synergetic effect and significantly inhibited bacterial growth. This result underscores the potential of VH43 to enhance the effectiveness of cephalosporins that are normally ineffective, offering a promising approach for combating bacterial infections.