Combined inactivation of the SOS response with TCA fumarases and the adaptive response enhances antibiotic susceptibility against Escherichia coli

Marina Murillo-Torres^1,2Isabel María Peñalver-Fernández¹Marta Quero-Delgado¹Sara Diaz-Diaz²María Romero-Muñoz¹Esther Recacha^2,3,4Fernando Docobo-Pérez^1,2,3José Manuel Rodriguez-Martínez^1,2,3*

• ¹Departamento de Microbiología, Facultad de Medicina, Universidad de Sevilla, Sevilla, Spain
• ²Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen Macarena / CSIC / Universidad de Sevilla, Sevilla, Spain
• ³Centro de Investigación Biomédica en Red en Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, Asturias, Spain
• ⁴Unidad Clínica de Enfermedades Infecciosas y Microbiología. Hospital Universitario Virgen Macarena, Sevilla, Spain

Targeting bacterial DNA damage responses such as the SOS response represents a promising strategy for enhancing the efficacy of existing antimicrobials. This study focuses on a recently discovered DNA damage response mechanism involving tricarboxylic acid cycle (TCA) fumarases and the adaptive response, crucial for Escherichia coli survival in the presence of genotoxic methyl methanesulfonate (MMS). We investigated whether this pathway contributes to protection against antibiotics, either separately or in combination with the SOS response.Methods: An isogenic collection of E. coli BW25113 mutants was used, including strains deficient in fumarases (ΔfumA, ΔfumB, ΔfumC) and the adaptive response (ΔalkA, ΔalkB, ΔaidB). Additional SOS response inactivation (ΔrecA) was conducted by P1 phage transduction. All mutants were subjected to antimicrobial susceptibility testing, growth curve analysis, survival and evolution assays. To validate the relevance of these findings, experiments were also performed in a quinolone-resistant E. coli ST131 clinical isolate.Results and discussion: Overall, no significant differences or only moderate increases in susceptibility were observed in the single mutants, with ΔfumC and ΔaidB mutants showing the highest susceptibility. To enhance this effect, these genes were then inactivated in combination with the SOS response by constructing ΔfumC/ΔrecA and ΔaidB/ΔrecA mutants. These combinations exhibited significant differences in susceptibility to various antimicrobials, particularly cephalosporins and quinolones, and especially in the ΔfumC/ΔrecA strain. To further assess these results, we constructed an E. coli ST131 ΔfumC/ΔrecA mutant, in which a similar trend was observed. Together, these findings suggest that co-targeting the SOS response together with fumarases or the adaptive response could enhance the effectiveness of antibiotics against E. coli, potentially leading to new therapeutic strategies.