Characterization of Nanobodies Targeting RecA: A Strategy for Bacterial SOS Response Inhibition

The global rise of antimicrobial resistance (AMR) presents a major threat to public health. A key contributor to AMR is the bacterial SOS response, a conserved DNA repair mechanism triggered by genotoxic stress. This pathway begins when the RecA recombinase detects single-stranded DNA, a marker of DNA damage, and forms the activated RecA* complex. RecA* then promotes the autocleavage of LexA, a transcriptional repressor, thereby inducing the expression of SOS genes. This response facilitates DNA repair, increases mutagenesis, and promotes horizontal gene transfer, all of which contribute to resistance. Inhibition of the SOS response offers a promising strategy to prevent the development and spread of antibiotic resistance. This thesis investigates the potential of nanobodies, single-domain antibody fragments derived from camelid heavy-chain antibodies, to selectively inhibit RecA and disrupt SOS pathway activation. In the study, a library of 125 nanobodies previously generated by immunizing llamas with RecA was analyzed. All of the nanobodies were first screened using a flow cytometry-based in vivo assay with the E. coli SMR6669 reporter strain, which expressed GFP after activation of the SOS response. Some of the most effective nanobodies from the screening were then produced in E. coli and purified. Three nanobodies, Nb21122, Nb21113, and Nb14523, were further tested in vitro using Fluorescence Polarization (FP) assays to evaluate their ability to block RecA oligomerization on single-stranded DNA (ssDNA) and prevent LexA from undergoing self-cleavage. Finally, the structure of the Nb-RecA complex involving Nb14523 was predicted using Boltz-2 and subjected to critical analysis.