Periodic Reporting for period 1 - ICECOUP (ICE coupling protein inhibitors)
Reporting period: 2024-03-15 to 2026-03-14
Antimicrobial Resistance (AMR) is one of the greatest threats to human health. Data from the European Centre for Disease Prevention and Control (ECDC) show that AMR is responsible for more than 35,000 deaths every year in the European Union/ European Economic area. The global overuse and misuse of antibiotics has resulted in the proliferation and dissemination of antibiotic resistant bacteria harbouring a multitude of Antibiotic Resistance (AR) genes that can be mobilized by different horizontal gene transfer processes. Conjugative plasmids and Integrative and Conjugative Elements (ICEs) constitute the main drivers of AR spread. Although ICEs outnumber conjugative plasmids, yet they still have been largely overlooked as vectors of AR and there is a substantial knowledge gap regarding their dissemination. ICEs are typically found integrated in the host chromosome and encode the machinery for their conjugation. Type IV Coupling proteins (T4CPs) are essential for conjugation, but only a few (mainly plasmid encoded) have been exhaustively studied. The main goal of ICECOUP was to in-depth characterize MobBICE, the T4CP of an ICE designated as ICEEc1 which was found in Escherichia coli ECOR31, with the aim of finding ICE conjugation inhibitors to block/minimise AR spread. The specific objectives of ICECOUP were: 1) To characterise MobBICE in vitro using different biochemical and biophysical techniques; 2) To assess MobBICE capacity to mobilise different mobile genetic elements (MGE) through different conjugation assays; and 3) To evaluate in vitro and in vivo potential of MobBICE inhibitors. Despite the early termination of the action, ICECOUP contributed to increasing the molecular knowledge about T4CPs belonging to ICEs of Gram-negative bacteria. Several molecular traits of MobBICE were described, as well as its interaction with other players involved in conjugation. In addition, it provided novel data about the interaction with mobilizable and conjugative plasmids.
A soluble version of MobBICE, lacking its Transmembrane Domains (TMDs), was purified and characterised. Its ability to interact with DNA was investigated by Electrophoretic Mobility Shift Assays with different DNA targets, but only a very weak interaction was detected. Its thermal stability was analysed in the presence of different concentrations of NaCl by Differential Scanning Fluorometry, showing that higher concentrations seemed to increase its stability. In addition, its ATP hydrolysis capacity was demonstrated by Malachite Green Phosphate Assay, and higher ATPase activity was observed in the presence of DNA.
Regarding the role of MobBICE in vivo, it was investigated through genetic engineering followed by conjugation assays. It was demonstrated that MobBICE interacts with the relaxase (another protein involved in conjugation) of ICEEc1 to transfer a plasmid that carries the origin of transference (oriT) of ICEEc1. The TMDs of MobBICE are required for this function. In addition, preliminary data about the interaction between the oriT of the ICEEc1 and one of the native plasmids of E. coli ECOR31 was shown. The ability of MobBICE to substitute other T4CPs, such as those from the mobilisable plasmid CloDF13 and the conjugative plasmid R388 was analysed. It was determined that they cannot substitute for each other.
Finally, the study of the effect of potential inhibitors of MobBICE was started. First, a positive control targeting a different protein of the conjugation system used, linoleic acid, was analysed. A decrease in the mobilisation frequency was observed, but no significant results were obtained. Then, dinoprost was evaluated, and surprisingly, although the results were not statistically significant, it seeemed to have the opposite effect to what was expected.
Regarding the role of MobBICE in vivo, it was investigated through genetic engineering followed by conjugation assays. It was demonstrated that MobBICE interacts with the relaxase (another protein involved in conjugation) of ICEEc1 to transfer a plasmid that carries the origin of transference (oriT) of ICEEc1. The TMDs of MobBICE are required for this function. In addition, preliminary data about the interaction between the oriT of the ICEEc1 and one of the native plasmids of E. coli ECOR31 was shown. The ability of MobBICE to substitute other T4CPs, such as those from the mobilisable plasmid CloDF13 and the conjugative plasmid R388 was analysed. It was determined that they cannot substitute for each other.
Finally, the study of the effect of potential inhibitors of MobBICE was started. First, a positive control targeting a different protein of the conjugation system used, linoleic acid, was analysed. A decrease in the mobilisation frequency was observed, but no significant results were obtained. Then, dinoprost was evaluated, and surprisingly, although the results were not statistically significant, it seeemed to have the opposite effect to what was expected.
ICEs have been largely overlooked as vectors of AR and they still remain poorly characterised. Most of the molecular understanding derives from the ICEs belonging to Gram-positive bacteria, and very few proteins implicated in ICE conjugation have been biochemical characterized. In ICECOUP, to the best of our knowledge, it is the first time that a T4CP belonging to an ICE from a Gram-negative bacterium has been purified and its ATPase enzymatic activity has been demonstrated. The characterisation of MobBICE is relevant for understanding Gram-negative bacteria horizontal gene transfer mechanisms, taking into account that its encoding gene, mobBICE, is present (more than 90% identity covering the whole sequence) in at least 2000 strains of Klebsiella pneumoniae, 200 of E. coli and other Enterobacteriae, according to genomic analysis.
Preliminary results of this project revealed that there is an interaction between the oriT of ICEEc1 and one of the conjugative plasmids present in the E. coli strain ECOR31. Nevertheless, no interaction was detected between the oriT, the relaxase and the T4CP of ICEEc1 and the equivalent elements of other MGE, such as the mobilisable plasmid CloDF13 or the conjugative plasmid R388. Defining the boundaries, characteristics and restrictions of these dynamic interactions is essential and, once more, underscores the necessity of experimental evidence besides genomic predictions to address complicated biological problems such as AR dissemination. A thorough characterization is also crucial for the identification and use of possible conjugation inhibitors. This project was also pioneer, as far as we know, in the use of a molecular component of an ICE as a target to control mobilization.
Overall, these results require further research to draw consistent conclusions. Nevertheless, ICECOUP has laid the foundation of a novel research line that will be continued within the host laboratory. In conclusion, this project has provided novel insights into the conjugation process of an ICE that in the long run can be used to design novel strategies to combat AR, such as the use of conjugation inhibitors targeting the T4CPs.
Preliminary results of this project revealed that there is an interaction between the oriT of ICEEc1 and one of the conjugative plasmids present in the E. coli strain ECOR31. Nevertheless, no interaction was detected between the oriT, the relaxase and the T4CP of ICEEc1 and the equivalent elements of other MGE, such as the mobilisable plasmid CloDF13 or the conjugative plasmid R388. Defining the boundaries, characteristics and restrictions of these dynamic interactions is essential and, once more, underscores the necessity of experimental evidence besides genomic predictions to address complicated biological problems such as AR dissemination. A thorough characterization is also crucial for the identification and use of possible conjugation inhibitors. This project was also pioneer, as far as we know, in the use of a molecular component of an ICE as a target to control mobilization.
Overall, these results require further research to draw consistent conclusions. Nevertheless, ICECOUP has laid the foundation of a novel research line that will be continued within the host laboratory. In conclusion, this project has provided novel insights into the conjugation process of an ICE that in the long run can be used to design novel strategies to combat AR, such as the use of conjugation inhibitors targeting the T4CPs.