2026-09-24T07:19:30Zhttps://iris.unica.it/oai/request

oai:iris.unica.it:11584/2667882022-10-15T19:58:14Zcom_11584_207615com_11584_111066col_11584_265854

Computational Study on MFP MexA in MexAB-OprM and RND transporter AcrB in AcrAB-TolC Efflux Pumps CACCIOTTO, PIERPAOLO ACRAB Efflux Pumps MD simulations OPRM TOLC biofisica biophysics escherichia coli gram-negative mexab multidrug resistance p. aeruginosa pompe di efflusso resistenza batterica salmonella Settore FIS/03 - Fisica della Materia In the last years we have assisted to the steady increase in the number of bacterial strains showing resistance to a wide variety of unrelated drugs, a phenomenon known as Multi-Drug Resistance (MDR). Molecular efflux pumps are among the major contributors to MDR, recognizing and expelling several noxious compounds into the external medium. In Gram-negative bacteria, efflux pumps of the Resistance Nodulation-cell Division (RND) superfamily are particularly relevant for the occurrence of MDR. These machineries are composed of a homotrimeric inner membrane drug/proton RND antiporter, a homotrimeric outer membrane factor (OMF), and an undetermined number of Membrane Fusion Proteins (MFPs), which are essential for the stability and functionality of the pumps. In our study we focused on the two major efflux pumps expressed in P.aeruginosa and E.coli / Salmonella, MexAB-OprM and AcrAB-TolC respectively. In particular, we studied the possible multimerization of the MFP component MexA and the effects of mutations in the AcrB distal binding pocket. The starting point for the first part of our study was the uncertainty about the number of MexA proteins involved in MexAB-OprM assembly. Recent studies suggested the possibility for MexA to assemble into multimers, i.e. dimers, trimers, etc. To investigate this possibility we studied the effects of the G72S mutation, which is known to impair the functionality of the efflux assembly presumably by affecting the multimerization of MexA. Extended MD simulation for wild type and mutant monomers showed large structural differences induced by the mutation. Furthermore, during the dimerization process (simulated with protein-protein docking) only for the wild type version we obtained potential dimeric units. New MD simulations confirmed the stability of such units and a convergence to a common structure. These results suggest that MexA dimerization is possible and the dimeric units are stable, within the simulation time. In the second part of the study we investigated the effects of G288D mutation in AcrB, identified during recent S.typhimurim infection. The AcrB variant showed an increased resistance to ciprofloxacin, suggesting a key role of the mutation in conferring the MDR. To study how the mutation, which is located in the distal binding pocket, might increase the efflux of ciprofloxacin we modeled AcrB structures for the wild type and the G288D variant. Reduced models, with only the periplasmic component, were then studied with MD simulations. The study revealed that the G288D substitution induced local structural changes in the AcrB distal binding pocket leading to a less compact structure with respect to the wild type. Furthermore, the mutation increased the polarity of this region suggesting different binding mechanisms that lead to a decreased susceptibility to ciprofloxacin and other fluoroquinolones. 2015-05-22 info:eu-repo/semantics/doctoralThesis http://hdl.handle.net/11584/266788 eng numberofpages:143 info:eu-repo/semantics/openAccess Università degli Studi di Cagliari license:Non specificato